ALK BREAK APART FISH
EVALUATION GUIDE
For NSCLC Tissue Specimens
Table of
Visualizing FISH Hybridization. . . . . . . . . . . . . . . . . . . 3
Slide Evaluation: Summary Procedure . . . . . . . . . . . . . 3
Contents Locate Target (Scribed) Area
Using 10x–25x Objective. . . . . . . . . . . . . . . . . . . . . . . . . . 4
Assess Target Area
Using 60x–100x Objective. . . . . . . . . . . . . . . . . . . . . . . . . 8
Selecting and Enumerating Cells
Within the Selected Target Area. . . . . . . . . . . . . . . . . . 16
Selecting Cells for Enumeration. . . . . . . . . . . . . . . . . . 17
Enumerating Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
ALK Enumeration (Signal Patterns). . . . . . . . . . . . . . . 19
ALK Status Determination. . . . . . . . . . . . . . . . . . . . . . . 21
All images property of Abbott Laboratories.
This guide provides an overview, examples and
useful tips for the evaluation of Vysis ALK Break
Apart FISH Probe Kit using fluorescence in situ
hybridization (FISH) on solid tumor formalin-fixed
paraffin-embedded (FFPE) non-small cell lung
cancer (NCSLC) tissue specimens.
VISUALIZING FISH HYBRIDIZATION
Evaluate hybridized FFPE NSCLC tissue specimen
slides using the recommended filter set on an
optimally performing fluorescence microscope.
SLIDE EVALUATION: SUMMARY PROCEDURE
The diagram below summarizes the process of slide evaluation using a fluorescent microscope.
LOCATE TARGET Use 10x to 25x objective to locate the area
VIEWING AREA of interest on the slide within the scribe mark
Use 60x to 100x objective to assess area
ASSESS TARGET AREA
for quality of signal and morphology
FIELD OF VIEW SELECTION
AND EVALUATION Scan scribed area to assess distribution of signals
CELL SELECTION Choose representative areas for enumeration
Using prescribed filters, enumerate 50 cells using
ENUMERATION
60x to 100x objective, record and calculate results
3
LOCATE TARGET (SCRIBED) AREA USING 10x–25x OBJECTIVE
Position the objective lens within the scribed area, to assure that only tumor cells are evaluated.
If necessary, use the H&E stained slide to confirm the target area prior to viewing the FISH slides.
NOTE: Accurate marking (scribing) of the tumor area is essential to avoid enumeration of adjacent,
non-tumor areas.
Tumor marked Tumor mark transferred
on H&E slide to the unstained slide
EXAMPLE 1. H&E stained tumor section: poorly differentiated adenocarcinoma. Figure A, overall
low-magnification view; Figure B, magnification of boxes 1 and 2.
Figure A.
NECROSIS
2
TUMOR CELLS
TUMOR STROMA
4
Figure B.
NECROSIS
NECROSIS
TUMOR CELLS
TUMOR CELLS
TUMOR CELLS
TUMOR CELLS
+ INFLAMMATORY CELLS
INFLAMMATORY CELLS
5
Use a 10x to 25x objective and the DAPI bandpass filter to locate the hybridized area of interest
on the slide within the scribe mark.
Choose areas where tumor cells are readily distinguishable:
• A
void areas of necrosis and where the nuclear borders are ambiguous (such as areas with
excess stroma)
• Skip nuclei with insufficient counterstain to determine the nuclear border
NOTE: Tumor specimens vary in appearance due to the specimen size and tumor biology, such
as histolopathologic type, pattern and grade. A 2013 guideline “Molecular Testing Guideline for
Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors” published by CAP,
IASLC and AMP, recommends users to become familiar with the morphological appearance of the
NSCLC specimens including the architectural features of the tumor. 2
EXAMPLE 2. Tumor areas acceptable for enumeration, with well-distinguishable tumor cells,
are encircled on 2 different tumor specimens. The specimen on the right has acceptable (indicated
by a circle) and unacceptable areas (indicated by an arrow) with tumor stroma and only a few
interspersed cells.
Lindeman N.I. et al. 2013. Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase
2
Inhibitors. Guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and
Association for Molecular Pathology. Arch. Pathol. Lab Med. 137(6):828–60.
6
EXAMPLE 3. Tumor areas not acceptable for enumeration.
Area with poorly distinguishable
tumor cells, no distinct cell boundaries
and clumps of cells observed.
Visible stroma and undigested
material between cells (pretreatment
or protease digestion may have
been insufficient).
7
ASSESS TARGET AREA USING 60x–100x OBJECTIVE
Using a 60x to 100x objective, use the prescribed filters to examine the quality of ALK signals and
quality of tissue morphology. Adjust the depth of the focus and become familiar with the size and
shape of the target signals and noise (debris). Verify that background appears dark and relatively
free of strong fluorescence that can make enumeration difficult.
EXAMPLE 4. FFPE slides viewed with four filters with the 60x objective.
DAPI DUAL O/G
GREEN ORANGE
8
ASSESS SLIDE HYBRIDIZATION ADEQUACY USING THE FOLLOWING CRITERIA:
• N
uclear morphology: Borders of tumor nuclei observed by DAPI should be distinguishable,
and nuclei should have good integrity.
EXAMPLE 5. Nuclear morphology evaluated with a 60x or 100x objective.
Nuclear morphology acceptable for signal enumeration
As visible with the DAPI filter,
the nuclear boundaries are easily
distinguished, nuclei have good
integrity and are not overlapping
(indicated with an arrow).
As visible in DAPI/orange/green,
the nuclear boundaries are easily
distinguished, nuclei have good
integrity and are not overlapping
(indicated with an arrow).
9
Nuclear morphology not acceptable for signal enumeration
Nuclear boundaries are not
distinguishable as visualized with the
DAPI filter. In this example, there is
an insufficient DAPI staining, and
nuclei are difficult to visualize.
Nuclear boundaries are not
distinguishable. In this example,
there is a visible undigested
material between cells that
obscures cell boundaries.
Nuclear boundaries are not
distinguishable. In this example,
high cell density resulted in
overlapping nuclei.
10
Nuclear boundaries are not
distinguishable and do not have good
integrity. In this example, nuclei
appear fragmented, DAPI staining is
weak due to tissue damage/loss.
Nuclear boundaries are not
distinguishable. DAPI staining
is weak. In this example, cellular
apoptosis with degradation of nuclear
morphology is suspected in the
selected area.
11
• B
ackground: The background should not contain particles that interfere with enumeration.
NOTE: In some cases, fluorescent haze or glow may be noticeable outside of the nuclei, but as long as the
fluorescent haze/glow does not cover the nuclei and interfere with the enumeration it is acceptable.
EXAMPLE 6. Fluorescent background
Fluorescent background acceptable for enumeration
Signals are bright, compact, and
well distinguishable against nuclear
background. Intercellular background
is dark, without particulates.
Fluorescent haze or glow is noticeable
outside of the nuclei, but does not
cover the nuclei and does not make
enumeration difficult.
12
Fluorescent background not acceptable for enumeration
Bright particulate fluorescent
background obscuring specific probe
signal (image captured with the dual
orange/green filter).
Hazy fluorescent background
obscures the signal (image captured
with green, above, and dual orange/
green, below, filters).
13
• P
robe signal intensity: The signals should be bright, distinct, and easily evaluable. Signals
should be compact, round or oval shapes. Overly diffuse signals should be avoided.
EXAMPLE 7. Probe signal intensity
Probe signal intensity acceptable for signal enumeration
Bright, distinct, and easily evaluable
fluorescent signals viewed with 100x
objective with the dual orange/green
filter. The signals are compact.
Bright, distinct, and easily evaluable
fluorescent signals viewed with 60x
objective with the dual orange and
green filters. The signals are compact.
14
Probe signal intensity not acceptable for signal enumeration
Weak signal intensity as viewed with
a triple (DAPI/orange/green) filter to
illustrate weak signal within nuclei
with distinct nuclear boundaries,
possibly due to the inappropriate
tissue fixation.
Weak/absent orange probe signal.
This specimen also shows indistinct
cell boundaries, damaged and irregular
cell morphology, possibly due to tissue
over-digestion.
Weak/absent green probe signal.
This specimen also shows indistinct
cell boundaries, damaged and irregular
cell morphology, and high nuclear
background, possibly due to tissue
over-digestion.
The majority of the target viewing areas selection should meet the quality criteria listed above.
Additionally, the target viewing areas must contain sufficient number of evaluable tumor cells.
15
SELECTING AND ENUMERATING CELLS
WITHIN THE SELECTED TARGET AREA
• S
elect an area of good nuclear distribution (i.e., where individual nuclei can be distinguished)
and ensure areas chosen for enumeration are representative of the signal distribution observed.
• U
sing a 60x to 100x objective and prescribed filters, begin analysis of the cells selected for
enumeration and record signals in each cell.
• M
ove to the next representative area (microscope field of view) for enumeration until 50 tumor
cells have been enumerated.
• F
ocus up and down to find all of the signals present in the nucleus. Enumerate the signals within
the nuclear boundary of each selected interphase tumor cell.
EXAMPLE 8. Relationship between tumor section, scribed target area, area chosen for
enumeration, and fields of view (microscope)
TUMOR SPECIMEN
SECTION
AREA CHOSEN FOR
ENUMERATION
MICROSCOPE
FIELDS OF VIEW
SCRIBE MARK
ENCIRCLING
TARGET AREA
16
SELECTING CELLS FOR ENUMERATION
EXAMPLE 9. Cell selection
Correct cell selection
Individual, non-overlapping nuclei
with distinct nuclear boundaries
are selected in this example (DAPI
filter image).
The selected cells have signals of
both colors, are free of fluorescent
background that obscures signals
(dual orange/green filter).
Incorrect cell selection
Overlapping cells in clumps with no
distinguishable nuclear boundaries.
Selected “cell” may contain several
overlapping cells and should not have
been selected.
17
Fluorescent particulate background
obscuring signal and interfering with
enumeration. Cells covered with the
particulate background should not
have been selected.
Weak signals and high background
interfering with enumeration. Signals
are diffuse, elongated. In the selected
cell, a hazy background obscures
probe signals, the signals are diffuse,
and nuclear boundaries are not
clearly distinguishable.
Cells indicated by circles contain
signal of only one color (green or
orange), and should not be selected
for enumeration. Cells selected for
enumeration must have at least one
signal of each color.
18
ENUMERATING CELLS
• U
sing a 60x to 100x objective and prescribed filters, begin analysis of the cells selected for
enumeration and record signals in each cell.
• F
ocus up and down to find all of the signals present in the nucleus. Enumerate the signals within
the nuclear boundary of each selected interphase tumor cell according to the enumeration
guidelines (see Enumeration section for example images), determining, for each cell, whether it
is positive or negative.
ALK ENUMERATION (SIGNAL PATTERNS)
EXAMPLE 10. Positive cells
These nuclei contain rearranged or “broken-apart” signals, 2 or more signal diameters apart.
The nucleus indicated by an
arrow has more than one set
of broken apart signals.
The nucleus indicated by an
arrow has one fused signal
(adjacent orange and green)
and one set of broken apart
signal individual green and
orange signal.
A nucleus can have a single
orange signal (deleted green
signal) in addition to fused
and/or broken apart signals.
In a nucleus indicated by an
arrow there are two fused signals
and one single orange signal.
19
The same nucleus may have
fused signals, broken apart
signals and/or deletions.
The positive cell indicated by
an arrow has one fused signal,
one broken-apart signal and
one single red signal. The
cell to the left has two fused
signals (negative cell).
This positive cell has two
fused signals and two
broken-apart signals.
EXAMPLE 11. Negative cells
Fused orange and green signals
are either overlapping, adjacent
or are less than two signal diameters
apart. If diffuse signals are adjacent
or connected by a fiber, they are
recorded as one fused signal.
In this example, a cell contains one
yellow fused signal, and adjacent
diffuse orange and green signals.
Multiple fused and/or broken
apart signals may be observed in
a single nucleus. This represents
tumor aneuploidy.
In this example, the cell indicated
by an arrow contains seven pairs
of fused signals. The adjacent cell
contains two pairs of fused signals.
20
A single green signal without
a corresponding orange signal
in addition to fused signal
(overlapping, adjacent, or are
less than 2 signal diameters
apart) is considered negative.
In this example, the cell with a
single green signal in addition
to two pairs of fused signals is
shown by an arrow.
NOTE: A nucleus with signals
of only one color should not
be enumerated. The cell shown
by an arrow has only orange
signals and should not be
selected for enumeration.
ALK STATUS DETERMINATION
• A sample is considered negative if < 5 cells out of 50 (< 5/50 or < 10%) are positive.
• A sample is considered positive if > 25 cells out of 50 (> 25/50 or > 50%) are positive.
• A
sample is considered equivocal if 5 to 25 cells (10 to 50%) are positive. If the sample is
equivocal, the slide is evaluated by the second reader who selects additional 50 nuclei
— T
he first and second cell count readings are added together and a percent is calculated
out of 100 cells (average percent of positive cells).
• If the average percent positive cells is < 15% (< 15/100), the sample is considered negative.
• If the average percent positive cells is ≥ 15% (≥ 15/100), the sample is considered positive.
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